CN102876805A - Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken - Google Patents
Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken Download PDFInfo
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Abstract
The invention discloses a primer system for PCR (polymerase chain reaction) identification for a deer, a pig, a cow, a sheep, a horse, a donkey, a rabbit and a chicken, and can detect eight animal species in one time so as to achieve a purpose that common animal species can be almost covered. The primer of the primer system only carries out specific amplification on a respective species target segment without reacting with other species, and therefore the primer can be suitable for multiple PCR reaction characterized in that at least two primer pairs are induced in one-time PCR reaction so as to effectively shorten experiment time. In addition, when the PCR is carried out, an optimized specific PCR reaction system and reaction program can be used, a detection flow is simplified due to the adoption of a uniform PCR detection method, a quick and efficient identification mode with the high specificity is established so as to effectively identify whether the deer blood product is true and false and identify the adulteration mode, and a modern molecular biology detection means is provided for deer blood quality control. In addition, the primer system can be cooperated with relevant reagent to be prepared into a kit, thereby being convenient to use. Meanwhile, possibility is provided for the industrial production and application, and the primer system has an excellent application prospect.
Description
Technical field
The present invention relates to biology field, the PCR that relates in particular to deer, pig, ox, sheep, horse, donkey, rabbit and chicken identifies with primer system and PCR authentication method, be applicable to rapid detection and the evaluation of above-mentioned eight kinds of animal blood goods, be particularly useful for the quick false proof evaluation of deer blood products.
Background technology
Deer blood is the blood of animal in deer family, puts down in writing according to Compendium of Material Medica: " the large tonifying deficiency of deer blood, benefiting essence-blood are separated the acne poison, medicine is malicious ".Deer blood has irreplaceability as traditional precious Chinese medicine, and its pharmaceutical use is approved extensively all the time and go deep into developing that product line is enriched constantly, comprises deer-blood wine, bright deer blood, Sanguis cervi crystal etc., and the marketable value that produces and demand are very huge.Under the ordering about of economic interests; the lawless person usually can utilize other common animals blood personation deer blood; or in the deer blood products, mix other common animals blood and adulterate to reduce composition, animal blood commonly used is the blood of pig, ox, sheep, horse, donkey, rabbit and chicken.But the Quality Detection of deer blood never has good means at present, and tradition range estimation and physico-chemical analysis method can't be differentiated the quite similar blood sample of these character effectively.In order to ensure validity and the purity of deer blood, from the quality of source control deer blood, exploitation is used for differentiating that the simple and rapid modern molecular biology detection technique of common species blood sample has urgency and necessity.
Polymerase chain reaction (PCR) technology has the advantage that response is quick, highly sensitive and specificity is good, it is the widest advanced element's biology techniques of current application, this technology utilizes special primer can amplify the dna fragmentation of respective specific from micro-example, reaches the purpose of amplifying nucleic acid signal.Simultaneously, PCR has very high atopic, can distinguish few difference to several nucleotide bases, so round pcr obtains application extensively and profoundly in the diagnostic assays field.Because there be conservative property and the polytropy on the sequence in the hereditary material DNA of different plant species during evolution, especially has higher conservative property in species inside, and there is larger otherness between the species.Utilize the above-mentioned high sensitivity of round pcr and specificity, can distinguish the different plant species material, especially have the product of higher economic worth.And the relative genomic dna of Mitochondrial DNA has higher mutation rate and hereditary polytropy, so the difference of mtdna sequence is usually used in evolutionary analysis and the diagnostic detection of different plant species and species inside.
Less for the document of above-mentioned report both at home and abroad at present, and mostly carry out for a small amount of species.Propagate such as artificial mad cow diseases that suppresses such as Dalmasso A, prevent from other products, mixing the ox goods, utilize animal component in the Multiplex PCR technical evaluation feed, used four pairs of primers respectively for ruminating animal, poultry, fish and pig, during design of primers for gene be: 12S rRNA, tRNA Val and 16S rRNA(Dalmasso A, Fontanella E, Piatti P, Civera T, Rosati S, Bottero MT, A multiplex PCR assay for the identification of animal species in feedstuffs. Mol. Cell. Probes, 2004,18:81-87.).Ghovvati, S. wait artificial the evaluation in factory's meat product whether to mingle, prevent from mixing pork product in the Islam food, designed respectively for ruminating animal, three pairs of primers of poultry and pig, during design of primers for gene be: 12S rRNA and 16S rRNA(Ghovvati, S., M.R. Nassiri, S.Z. Mirhoseini, A.H. Moussavi, A. Javadmanes, Fraud identification in industrial meat products by multiplex PCR assay. Food Control, 2009,20:696-699.).The artificial situation of mingling that detects in the deer product such as Dai-Ming Zha and for example, four pairs have been designed respectively for the primer of ox, sheep, poultry and pig, during design of primers for gene be: tRNA Val and 16S rRNA(Dai-Ming Zha, Xiu-Mei Xing, Fu-He Yang, A multiplex PCR assay for fraud identification of deer products, Food Control, 2010,21:1402-1407.).Therefore but such scheme does not design the primer for deer, although can whether contain this four classes material in the working sample, whether can not determine the deer product.
Can find out that by above representative result of study there is following deficiency in these detections: (1) sensing range is narrow, once can only detect a small amount of species, substantially can be greater than four.This is because in same total dna profiling system, and the right quantity of primer is more, and primer pair is more serious with the phase mutual interference between total dna profiling, and the specificity that PCR reacts also more can not guarantee; (2) foundation is not to quick and high primer system and the detection method of specificity of a large amount of common species, and this also can cause undetected phenomenon.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the PCR that the invention provides a kind of deer pig, cattle and sheep horse donkey rabbit chicken identifies the primer system of using, this primer system and the authentication method that carries out with this primer system can be fast and the true and false of the high detection deer pig, cattle and sheep horse donkey rabbit chicken blood goods of specificity and whether having mingle, be particularly useful for the deer blood products and identify and detection that its detection coverage rate to common species is extensive.
The present invention for the technical scheme that solves its technical problem and adopt is:
The PCR of a kind of deer pig, cattle and sheep horse donkey rabbit chicken identifies and uses the primer system, by following primer to forming:
(1) deer 12S rRNA gene is increased the primer that generates deer specific amplification fragment to I:
Forward primer: 5 '-GCCTTCCTATTGACCCTTAATAG-3 '
Reverse primer: 5 '-AGCGGCTGTAAAGTTACTTTCGT-3 ';
(2) pig 12S rRNA gene is increased the primer that generates pig specific amplification fragment to II:
Forward primer: 5 '-AGCACACCTATAACGGTAGCTCA-3 '
Reverse primer: 5 '-CCTAGCTATCGTGTGTCAGGATT-3 ';
(3) ox Cytb gene is increased the primer that generates Newt opposite sex amplified fragments to III:
Forward primer: 5 '-CCCTCTTACTAATTCTAGCTCT-3 '
Reverse primer: 5 '-GTCCGATGGTGATATATGGGTG-3 ';
(4) sheep Cytb gene is increased the primer that generates sheep specific amplification fragment to IV:
Forward primer: 5 '-CGCCATGCTACTAATTCTTGTTCT-3 '
Reverse primer: 5 '-TGGAGGAAGGGTACAAGTACTAAG-3 ';
(5) to the horse gene increase generate horse specific amplification fragment primer to combination, this primer generates horse specific amplification fragment by horse Cytb gene is increased to combination primer forms VI to V with to the horse 12S rRNA gene primer that generates horse specific amplification fragment that increases:
Primer is to V:
Forward primer: 5 '-CACAGCCCTGGTAGTCGTACA-3 '
Reverse primer: 5 '-TGTCCGCCGATTCATGTTAGT-3 ';
Primer is to VI:
Forward primer: 5 '-CGTGTCAAAGACTAATACCAA-3 '
Reverse primer: 5 '-AGCTATCGTGTAGTCAGAGGTA-3 ';
(6) donkey 16S rRNA gene is increased the primer that generates donkey specific amplification fragment to VII:
Forward primer: 5 '-TTCAAGCTCAACGTCACACATA-3 '
Reverse primer: 5 '-TGTGTTGGGTTAACAATAGTCT-3 ';
(7) rabbit 12S rRNA gene is increased the primer that generates rabbit specific amplification fragment to VIII:
Forward primer: 5 '-GCGTAAAGCGTGATTAGAATAAAC-3 '
Reverse primer: 5 '-TAGCTATCGTGAGTTCGAAGAGT-3 ';
(8) chicken 12S rRNA gene is increased the primer that generates the specific chicken amplified fragments to IX:
Forward primer: 5 '-ATCCATCTTAGCCTCAACGATTAA-3 '
Reverse primer: 5 '-CTATTGAGCTCACTGTTGTTCTTT-3 '.
The present invention is by analyzing the heredity conservative region gene (comprising 12S rRNA, 16S rRNA and Cytb) not in deer, pig, ox, sheep, horse, donkey, rabbit and eight kinds of animal mitochondria DNAs of chicken, after carrying out sequencing and comparison, designed respectively the specific PCR primer pair of each species according to the special base site on each sequence, it only has amplified signal to the total dna profiling of corresponding species, other species are not had the purpose amplified signal, therefore can carry out qualitative detection to each species fast and accurately.Primer of the present invention is to all well known to a person skilled in the art that chemical synthesis process carries out the DNA synthetic technology and obtains by utilization.
The present invention also provides the PCR authentication method of a kind of deer pig, cattle and sheep horse donkey rabbit chicken to be, take sample total DNA as template, utilize described primer to I, primer to II, primer to III, primer to IV, by primer V and primer are respectively carried out pcr amplification experiment to VIII and primer to IX to VII, primer to combination, primer to the primer that VI forms, after reaction finishes according to the agarose gel electrophoresis result of determination; Described sample total DNA template is from the mixing of deer, pig, ox, sheep, horse, donkey, rabbit and chicken species blood, or a kind of in above-mentioned each species blood products.
Specific PCR reaction system in the experiment of described pcr amplification comprises following component: dNTPs 0.8 parts by volume of distilled water 3 parts by volume, 10 * PCR reaction buffer, 1 parts by volume, 2.5mM each, Taq enzyme 0.2 parts by volume of 2.5U/ μ l, through diluting 500 times total dna profiling 1 parts by volume, 1 μ M forward primer 2 parts by volume and 1 μ M reverse primer, 2 parts by volume.Its optimal component is the Taq enzyme 0.2 μ l of dNTPs 0.8 μ l, the 2.5U/ μ l of distilled water 3 μ l, 10 * PCR reaction buffer, 1 μ l, 2.5mM each, through diluting 500 times total dna profiling 1 μ l, 1 μ M forward primer, 2 μ l and 1 μ M reverse primer, 2 μ l, amount to 10 μ l.
Specific PCR response procedures is in the described pcr amplification experiment: carry out successively 94 ℃ of lower denaturation 5min, and 94 ℃ of sex change 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 45s, carry out 35 times and circulate, and 72 ℃ of downward-extension 10min finish behind the 1min 4 ℃ of lower maintenances at last.
Carry out in the authentication method of the present invention agarose gel electrophoresis finish after during result of determination, judgment basis be described primer to I, primer to II, primer to III, primer to IV, big or small to the dna fragmentation that IX amplifies respectively to VIII and primer to VII, primer to combination, primer to the primer that VI forms to V and primer by primer; Wherein
The primer of deer species is 376bp to the dna fragmentation size that I amplifies;
The primer of pig species is 300bp to the dna fragmentation size that II amplifies;
The primer of ox species is 360bp to the dna fragmentation size that III amplifies;
The primer of sheep species is 231bp to the dna fragmentation size that IV amplifies;
The dna fragmentation size that the primer that V and primer is made of VI primer of horse species amplifies combination is for corresponding to respectively 456bp and 115bp;
The primer of donkey species is 232bp to the dna fragmentation size that VII amplifies;
The primer of rabbit species is 126bp to the dna fragmentation size that VIII amplifies;
The primer of chicken species is 299bp to the dna fragmentation size that IX amplifies.
The present invention also provides the PCR identifier box of a kind of deer pig, cattle and sheep horse donkey rabbit chicken, comprise primer that described primer forms VI V and primer to IV, by primer III, primer II, primer I, primer to combination, primer to VII, primer to VIII and primer to IX.Primer system of the present invention and related reagent can be assembled into test kit, use with convenient.Wherein said related reagent can be other reagent except total dna profiling in the specific PCR reaction system described in the present invention; Also can be reagent except the sample total DNA template that other are applicable, as being used for some conventional reagent of PCR reaction, or be tested the composition etc. of the conventional reagent that obtains through limited number of time by those skilled in the art.Also include in the test kit of the present invention in addition and implement basic apparatus required for the present invention.
Test kit of the present invention is formed by a plurality of partition, in order to hold fixing one or more container such as pipe or bottle class.The primer pair of each species among the present invention can be housed respectively in these containers, also the primer of each species can be loaded on wherein after to mixture being mixed into according to a certain percentage primer, according to actual needs the primer of each species to or primer can be lyophilized form to mixture or be dissolved in state in the aforementioned related reagent.
Test kit of the present invention is used for the species evaluation that the total dna profiling of single species carries out deer, pig, ox, sheep, horse, donkey, rabbit and chicken.And described test kit is used for many species and mixes the species evaluation that total dna profiling carries out deer, pig, ox, sheep, horse, donkey, rabbit and chicken.Described test kit is particularly useful for the true and false of deer blood products and mingles judgement.
Useful technique effect of the present invention is: the primer system of the application of the invention, can disposable detection reach eight animal species, and reach the purpose that the common animals species are covered substantially; And primer of the present invention all only carries out specific amplification to the target fragment of species separately, can not increase other the zone and can not react with other species, therefore also can be applicable to import the right multi-PRC reaction of two or more primer in the PCR reaction, thereby can effectively shorten experimental period; This is external when carrying out the PCR reaction, use specific PCR reaction system and the response procedures optimized, adopt unified PCR detection method, simplified testing process, set up rapidly and efficiently and the high evaluation mode of specificity, thereby effectively identify the true and false of deer blood product and mingle situation, the detection means of modern molecular biology is provided for the quality control of deer blood; This primer system can also cooperate related reagent generate a reagent box in addition, the convenient use, simultaneously for suitability for industrialized production and application provide may, its application prospect is fabulous.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of total dna profiling of extracting of eight kinds of animal bloods of the present invention;
Fig. 2 is that the primer with deer 12S rRNA gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to I as primer;
Fig. 3 is that the primer with pig 12S rRNA gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to II as primer;
Fig. 4 is that the primer with ox Cytb gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to III as primer;
Fig. 5 is that the primer with sheep Cytb gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to IV as primer;
Fig. 6 is two pairs of Auele Specific Primers with horse of the present invention, and namely the Auele Specific Primer of horse Cytb gene carries out pcr amplification gained agarose gel electrophoresis figure as primer to the Auele Specific Primer of V and horse 12S rRNA gene jointly to VI;
Fig. 7 is that the primer with donkey 16S rRNA gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to VII as primer;
Fig. 8 is that the primer with rabbit 12S rRNA gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to VIII as primer;
Fig. 9 is that the primer with chicken 12S rRNA gene of the present invention carries out pcr amplification gained agarose gel electrophoresis figure to IX as primer;
Figure 10 be take the total DNA sample of each species of the present invention by with the total DNA sample of the mixed mixing of volume as template, use each Species-specific primer to mix therewith respectively total dna profiling and carry out pcr amplification gained agarose gel electrophoresis figure;
Wherein M representative standard is adjusted the molecular weight label, and this label from up to down is followed successively by 2kb, 1kb, 750bp, 500bp, 250bp and 100bp among Fig. 1; This label from up to down is followed successively by 500bp, 400bp, 350bp, 300bp, 250bp, 200bp, 150bp, 100bp and 50bp among Fig. 2 to Figure 10; Wherein number 1 ~ 8 among Fig. 1 to Figure 10 and represent successively deer, pig, ox, sheep, horse, donkey, rabbit and chicken.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
One, the design of each Species-specific primer:
For Mitochondrial DNA more not conservative on evolving, utilize the U.S. state-run biotechnology information center (NCBI) GenBank, to deer, pig, ox, sheep, horse, donkey, eight common species of rabbit and chicken carry out determined dna sequence and compare of analysis, filter out the changeable specific target areas of heredity, comprise 12S rRNA, 3 genes of 16S rRNA and Cytb, designed respectively the specific PCR primer pair of each species for the specificity base site on each sequence of said gene, and compare online through NCBI BLAST database, further confirm the species specificity of primer, be used for pcr amplification purpose fragment.
Each primer is as described below:
Deer 12S rRNA gene is increased the primer that generates deer specific amplification fragment to I:
Forward primer: 5 '-GCCTTCCTATTGACCCTTAATAG-3 ' (SEQ ID NO.1)
Reverse primer: 5 '-AGCGGCTGTAAAGTTACTTTCGT-3 ' (SEQ ID NO.2);
Pig 12S rRNA gene is increased the primer that generates pig specific amplification fragment to II:
Forward primer: 5 '-AGCACACCTATAACGGTAGCTCA-3 ' (SEQ ID NO.3)
Reverse primer: 5 '-CCTAGCTATCGTGTGTCAGGATT-3 ' (SEQ ID NO.4);
Ox Cytb gene is increased the primer that generates Newt opposite sex amplified fragments to III:
Forward primer: 5 '-CCCTCTTACTAATTCTAGCTCT-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-GTCCGATGGTGATATATGGGTG-3 ' (SEQ ID NO.6);
Sheep Cytb gene is increased the primer that generates sheep specific amplification fragment to IV:
Forward primer: 5 '-CGCCATGCTACTAATTCTTGTTCT-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-TGGAGGAAGGGTACAAGTACTAAG-3 ' (SEQ ID NO.8);
To the horse gene increase generate horse specific amplification fragment primer to combination, this primer generates horse specific amplification fragment by horse Cytb gene is increased to combination primer forms VI to V with to the horse 12S rRNA gene primer that generates horse specific amplification fragment that increases:
Primer is to V:
Forward primer: 5 '-CACAGCCCTGGTAGTCGTACA-3 ' (SEQ ID NO.9)
Reverse primer: 5 '-TGTCCGCCGATTCATGTTAGT-3 ' (SEQ ID NO.10);
Primer is to VI:
Forward primer: 5 '-CGTGTCAAAGACTAATACCAA-3 ' (SEQ ID NO.11)
Reverse primer: 5 '-AGCTATCGTGTAGTCAGAGGTA-3 ' (SEQ ID NO.12);
Donkey 16S rRNA gene is increased the primer that generates donkey specific amplification fragment to VII:
Forward primer: 5 '-TTCAAGCTCAACGTCACACATA-3 ' (SEQ ID NO.13)
Reverse primer: 5 '-TGTGTTGGGTTAACAATAGTCT-3 ' (SEQ ID NO.14);
Rabbit 12S rRNA gene is increased the primer that generates rabbit specific amplification fragment to VIII:
Forward primer: 5 '-GCGTAAAGCGTGATTAGAATAAAC-3 ' (SEQ ID NO.15)
Reverse primer: 5 '-TAGCTATCGTGAGTTCGAAGAGT-3 ' (SEQ ID NO.16);
Chicken 12S rRNA gene is increased the primer that generates the specific chicken amplified fragments to IX:
Forward primer: 5 '-ATCCATCTTAGCCTCAACGATTAA-3 ' (SEQ ID NO.17)
Reverse primer: 5 '-CTATTGAGCTCACTGTTGTTCTTT-3 ' (SEQ ID NO.18).
Two, the extracting of the total DNA of each species blood:
The blood collection of above-mentioned eight kinds of animals uses anti-freezing test tube packing (1ml/ pipe) in the healthy animal live body, and is frozen in-80 ℃, carries out putting to room temperature before the total DNA extracting of blood.The total DNA(of each animal species blood is comprising Mitochondrial DNA) extracting all adopt QIAGEN company QIAamp DNA Mini and Blood Mini test kit to carry out, its extraction steps is as described below:
(1) draws 20 μ l proteolytic enzyme (Protease) to 1.5ml centrifuge tube bottom;
(2) add a kind of in above-mentioned eight kinds of animal blood sample that 200 μ l gather, softly blow and beat mixing; Add 200 μ l AL damping fluids, some shake mixing 15s;
(3) after 10 minutes, carry out of short duration centrifugal at 56 ℃ of incubations;
(4) add some shake mixing 15s behind the 200 μ l dehydrated alcohols, then carry out of short duration centrifugally, obtain mixed solution;
(5) in advance micro-column spinner (Mini spin column) is placed on the 2ml collection tube, then gained mixed solution in (4) is moved into Mini spin column, then centrifugal 1min under 12000r/min, centrifugal end places Mini spin column on the new 2ml collection tube;
(6) open the lid of Mini spin column, add the damping fluid of 500 μ l AW1, then under 12000r/min, behind the centrifugal 1min, Mini spin column is placed on the new 2ml collection tube;
(7) open the lid of Mini spin column, add after the 500 μ l AW2 damping fluids centrifugal 1min under 16000r/min;
(8) Mini spin column is placed on the new 1.5ml collection tube that cuts off lid centrifugal 2min under 16000r/min; Then Mini spin column is placed again on the new 1.5ml collection tube that cuts off lid, add 200 μ l AE damping fluids, after room temperature leaves standstill 1min, centrifugal 1min under 12000r/min; Total DNA sample that will elute at last is frozen in-20 ℃.
Whether the total DNA by each animal species of above-mentioned steps extracting gained can be used for further amplification experiment, need to judge by the agarose gel electrophoresis experiment, if obtain dyeing clearly band, illustrate that then the total DNA of extracting gained can be used for further amplification experiment.Among the present invention the total DNA sample of each species of extracting gained carried out the experiment of 1wt.% agarose gel electrophoresis, and in gel, add ethidium bromide (EB) and dye.Adopted the 120V electrophoresis 40 minutes, when tetrabromophenol sulfonphthalein band migration stop electrophoresis, then gel imaging during to the gel edge.Wherein each well applied sample amount is 20 μ l.
Acquired results as shown in Figure 1.As seen from Figure 1, total DNA sample of each animal blood extracting gained of the present invention obtains gem-pure band behind electrophoresis dying, there is no the unclear situation of disperse, the interpret sample Mitochondrial DNA is complete without degraded, and quality is fine to can be used for further PCR experiment.
Three, each Species-specific primer is to carrying out the PCR experiment:
Adopt the Auele Specific Primer of synthetic each animal species of gained in the step 1 to carrying out the PCR experiment, selected total dna profiling can be total DNA sample of each animal species in the step 2, also can be for the total DNA sample of by a certain percentage mixed mixing of the total DNA sample of each animal species of gained in the step 2.In the present invention, for the total DNA sample of described each species with mix total DNA sample and all under same PCR reactive system and same PCR response procedures, carry out.Specific as follows described.
Embodiment one:
Take the total DNA sample of described each species as total dna profiling:
(1) processing of the total DNA sample of each species: total DNA sample of described eight kinds of animals is respectively got behind the 1 μ l 500 times of dilutions, as total dna profiling, and primer is diluted to 1 μ mol/L;
(2) preparation of reaction system: in the PCR thin-walled tube, add following each reactive component, reaction system 10 μ l/ pipe.
(3) formulation of PCR response procedures: carry out successively 94 ℃ of lower denaturation 5min, 94 ℃ of sex change 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 45s, carry out 35 circulations, and 72 ℃ of downward-extension 10min finish behind 4 ℃ of lower maintenance 1min at last, get the PCR reaction product.
(4) 2wt.% agarose gel electrophoresis: gained PCR reaction product in (3) is carried out the 2wt.% agarose gel electrophoresis, adding ethidium bromide (EB) in the gel dyes, and then electrophoresis 40 minutes under the 120V, when tetrabromophenol sulfonphthalein band migration stop electrophoresis, then gel imaging during to the gel edge.
Gained gel imaging result such as Fig. 2 are to shown in Figure 9.
Wherein Fig. 2 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of deer 12S rRNA gene I is carried out the DNA band that pcr amplification obtains as primer.The result shows that deer species special primer only goes out the band of 376bp size to I to the total dna profiling specific amplified of deer blood, and other species dna profiling is without amplification.
Fig. 3 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of pig 12S rRNA gene II is carried out the DNA band that pcr amplification obtains as primer.The result shows that pig species special primer only goes out the band of 300bp size to II to the total dna profiling specific amplified of pig blood, and other species dna profiling is without amplification.
Fig. 4 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of ox Cytb gene III is carried out the DNA band that pcr amplification obtains as primer.The result shows that ox species special primer only goes out the band of 360bp size to III to the total dna profiling specific amplified of ox blood, and other species dna profiling is without amplification.
Fig. 5 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of sheep Cytb gene IV is carried out the DNA band that pcr amplification obtains as primer.The result shows that sheep species special primer only goes out the band of 231bp size to IV to the total dna profiling specific amplified of sheep blood, and other species dna profiling is without amplification.
Fig. 6 is the total dna profiling that adopts respectively eight kinds of animals, and with two pairs of Auele Specific Primers of horse, namely the Auele Specific Primer of horse Cytb gene carries out the DNA band that pcr amplification obtains as primer to the Auele Specific Primer of V and horse 12S rRNA gene jointly to VI.The result shows that the Auele Specific Primer of above-mentioned two pairs of horse species has only gone out two bands of 456bp and 115bp size to the total dna profiling specific amplified of horse blood, and to other species dna profilings without amplification.
Fig. 7 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of donkey 16S rRNA gene VII is carried out the DNA band that pcr amplification obtains as primer.The result shows that donkey species special primer only goes out the band of 232bp size to VII to the total dna profiling specific amplified of donkey blood, and other species dna profiling is without amplification.
Fig. 8 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of rabbit 12S rRNA gene VIII is carried out the DNA band that pcr amplification obtains as primer.The result shows that rabbit species special primer only goes out the band of 126bp size to VIII to the total dna profiling specific amplified of rabbit blood, and other species dna profiling is without amplification.
Fig. 9 is the total dna profiling that adopts respectively eight kinds of animals, with the primer of chicken 12S rRNA gene IX is carried out the DNA band that pcr amplification obtains as primer.The result shows that chicken species special primer only goes out the band of 299bp size to IX to the total dna profiling specific amplified of chicken blood, and other species dna profiling is without amplification.
By above-mentioned experiment and experimental result as can be known, when the list of above-mentioned each species carries out the PCR experiment with eight kinds of total dna profilings respectively to primer (horse is two pairs of primers), determine that this primer only responds to total dna profiling of himself species, and total dna profiling of other seven species is not reacted, verified the specificity of each species primer in single species dna profiling mode.
Embodiment two:
Take the total DNA sample of according to a certain percentage mixed mixing of the total DNA sample of described each species as template:
(1) processing of the total DNA sample of mixing: total DNA sample equal-volume of described eight kinds of animals is mixed, and wherein every kind accounts for 12.5vol.%, and gained is for mixing total DNA sample.Get 1 μ l and should mix total DNA sample and dilute 500 times, as mixing total dna profiling, and primer is diluted to 1 μ mol/L;
(2) preparation of reaction system: in the PCR thin-walled tube, add following each reactive component, reaction system 10 μ l/ pipe.
Mix total dna profiling 1 μ l, 1 μ M forward primer, 2 μ l and 1 μ M reverse primer, 2 μ l described in the Taq enzyme 0.2 μ l of dNTPs 0.8 μ l, the 2.5U/ μ l of distilled water 3 μ l, 10 * PCR reaction buffer, 1 μ l, 2.5mM each, (1); Wherein the Taq enzyme is conventional Taq enzyme (referring to the Taq enzyme specification sheets of Tiangen company).
(3) formulation of PCR response procedures: this response procedures is with the response procedures among the embodiment one.
(4) 2wt.% agarose gel electrophoresis: this electrophoresis process and condition are with the electrophoresis among the embodiment one.
Gained gel imaging result as shown in figure 10.The method with each Species-specific primer respectively with mix total dna profiling and carry out pcr amplification reaction, well verified validity and the specificity of each Species-specific primer under complicated total dna profiling condition.As seen from Figure 10, each Species-specific primer all can well amplify the band of the specificity size of its corresponding species from total DNA hybrid template.
According to the PCR reaction system described in above-described embodiment one and the embodiment two, primer system of the present invention can cooperate related reagent to be assembled into the test kit of various combination, can be used for deer, pig, ox, sheep, horse, donkey, rabbit and chicken species mirror
Fixed, be particularly useful for for the true and false of deer blood products and mingle judgement.
Claims (10)
1. the PCR of a deer pig, cattle and sheep horse donkey rabbit chicken identifies and to use the primer system, it is characterized in that: by following primer to forming:
(1) deer 12S rRNA gene is increased the primer that generates deer specific amplification fragment to I:
Forward primer: 5 '-GCCTTCCTATTGACCCTTAATAG-3 '
Reverse primer: 5 '-AGCGGCTGTAAAGTTACTTTCGT-3 ';
(2) pig 12S rRNA gene is increased the primer that generates pig specific amplification fragment to II:
Forward primer: 5 '-AGCACACCTATAACGGTAGCTCA-3 '
Reverse primer: 5 '-CCTAGCTATCGTGTGTCAGGATT-3 ';
(3) ox Cytb gene is increased the primer that generates Newt opposite sex amplified fragments to III:
Forward primer: 5 '-CCCTCTTACTAATTCTAGCTCT-3 '
Reverse primer: 5 '-GTCCGATGGTGATATATGGGTG-3 ';
(4) sheep Cytb gene is increased the primer that generates sheep specific amplification fragment to IV:
Forward primer: 5 '-CGCCATGCTACTAATTCTTGTTCT-3 '
Reverse primer: 5 '-TGGAGGAAGGGTACAAGTACTAAG-3 ';
(5) to the horse gene increase generate horse specific amplification fragment primer to combination, this primer generates horse specific amplification fragment by horse Cytb gene is increased to combination primer forms VI to V with to the horse 12S rRNA gene primer that generates horse specific amplification fragment that increases:
Primer is to V:
Forward primer: 5 '-CACAGCCCTGGTAGTCGTACA-3 '
Reverse primer: 5 '-TGTCCGCCGATTCATGTTAGT-3 ';
Primer is to VI:
Forward primer: 5 '-CGTGTCAAAGACTAATACCAA-3 '
Reverse primer: 5 '-AGCTATCGTGTAGTCAGAGGTA-3 ';
(6) donkey 16S rRNA gene is increased the primer that generates donkey specific amplification fragment to VII:
Forward primer: 5 '-TTCAAGCTCAACGTCACACATA-3 '
Reverse primer: 5 '-TGTGTTGGGTTAACAATAGTCT-3 ';
(7) rabbit 12S rRNA gene is increased the primer that generates rabbit specific amplification fragment to VIII:
Forward primer: 5 '-GCGTAAAGCGTGATTAGAATAAAC-3 '
Reverse primer: 5 '-TAGCTATCGTGAGTTCGAAGAGT-3 ';
(8) chicken 12S rRNA gene is increased the primer that generates the specific chicken amplified fragments to IX:
Forward primer: 5 '-ATCCATCTTAGCCTCAACGATTAA-3 '
Reverse primer: 5 '-CTATTGAGCTCACTGTTGTTCTTT-3 '.
2. the PCR authentication method of a deer pig, cattle and sheep horse donkey rabbit chicken, it is characterized in that: take sample total DNA as template, utilize described primer to I, primer to II, primer to III, primer to IV, by primer V and primer are respectively carried out pcr amplification experiment to VIII and primer to IX to VII, primer to combination, primer to the primer that VI forms, after reaction finishes according to the agarose gel electrophoresis result of determination; Described sample total DNA template is from the mixing of deer, pig, ox, sheep, horse, donkey, rabbit and chicken species blood, or a kind of in above-mentioned each species blood products.
3. the PCR authentication method of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 2 is characterized in that: the specific PCR reaction system in the described pcr amplification experiment comprises following component: dNTPs 0.8 parts by volume of distilled water 3 parts by volume, 10 * PCR reaction buffer, 1 parts by volume, 2.5mM each, Taq enzyme 0.2 parts by volume of 2.5U/ μ l, through diluting 500 times total dna profiling 1 parts by volume, 1 μ M forward primer 2 parts by volume and 1 μ M reverse primer, 2 parts by volume.
4. the PCR authentication method of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 3 is characterized in that: the specific PCR reaction system in the described pcr amplification experiment comprises following component: the Taq enzyme 0.2 μ l of dNTPs 0.8 μ l, the 2.5U/ μ l of distilled water 3 μ l, 10 * PCR reaction buffer, 1 μ l, 2.5mM each, through diluting 500 times total dna profiling 1 μ l, 1 μ M forward primer, 2 μ l and 1 μ M reverse primer, 2 μ l.
5. the PCR authentication method of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 2, it is characterized in that: specific PCR response procedures is in the described pcr amplification experiment: carry out successively 94 ℃ of lower denaturation 5min, 94 ℃ of sex change 45s, 56 ℃ of annealing 45s, 72 ℃ are extended 45s, carry out 35 circulations, 72 ℃ of downward-extension 10min finish behind 4 ℃ of lower maintenance 1min at last.
6. according to claim 2 to the PCR authentication methods of 5 described deer pig, cattle and sheep horse donkey rabbit chickens, it is characterized in that: after described agarose gel electrophoresis finishes during result of determination, judgment basis be described primer to I, primer to II, primer to III, primer to IV, big or small to the dna fragmentation that IX amplifies respectively to VIII and primer to VII, primer to combination, primer to the primer that VI forms to V and primer by primer; Wherein
The primer of deer species is 376bp to the dna fragmentation size that I amplifies;
The primer of pig species is 300bp to the dna fragmentation size that II amplifies;
The primer of ox species is 360bp to the dna fragmentation size that III amplifies;
The primer of sheep species is 231bp to the dna fragmentation size that IV amplifies;
The dna fragmentation size that the primer that V and primer is made of VI primer of horse species amplifies combination is for corresponding to respectively 456bp and 115bp;
The primer of donkey species is 232bp to the dna fragmentation size that VII amplifies;
The primer of rabbit species is 126bp to the dna fragmentation size that VIII amplifies;
The primer of chicken species is 299bp to the dna fragmentation size that IX amplifies.
7. the PCR identifier box of a deer pig, cattle and sheep horse donkey rabbit chicken is characterized in that: comprise primer that described primer forms VI V and primer to IV, by primer III, primer II, primer I, primer to combination, primer to VII, primer to VIII and primer to IX.
8. the PCR identifier box of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 7 is characterized in that: described test kit is used for the species that the total dna profiling of single species carries out deer, pig, ox, sheep, horse, donkey, rabbit and chicken to be identified.
9. the PCR identifier box of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 7 is characterized in that: described test kit is used for many species and mixes the species that total dna profiling carries out deer, pig, ox, sheep, horse, donkey, rabbit and chicken and identify.
10. the PCR identifier box of deer pig, cattle and sheep horse donkey rabbit chicken according to claim 7 is characterized in that: described test kit is used for the true and false of deer blood products and mingles judgement.
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